Topical ophthalmic compositions containing tobramycin and dexamethasone

ABSTRACT

Ophthalmic pharmaceutical compositions containing tobramycin, dexamethasone and deacetylated xanthan gum are described. The compositions provide longer ocular retention for enhanced ocular bioavailability of tobramycin and dexamethasone. In a preferred embodiment, the compositions also provide for improved suspension of dexamethasone. The concentration of ionizable species in the compositions is controlled so as to prevent precipitation of the xanthan gum as a result of ionic interactions between tobramycin and xanthan gum, while allowing for a restoration of viscosity upon topical application of the compositions to the eye. The use of deacetylated xanthan gum is disclosed, so as to avoid formulation instability caused by pH drift during storage.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 12/841,414 filed Jul. 22, 2010, which is a divisional of U.S.patent application Ser. No. 11/960,196 filed Dec. 19, 2007 (now U.S.Pat. No. 7,795,316).

BACKGROUND OF THE INVENTION

The present invention is directed to the field of ophthalmicanti-infective/anti-inflammatory compositions and associated methods oftreatment in mammals, particularly humans. More specifically, thepresent invention is directed to new ocularanti-infective/anti-inflammatory compositions containing tobramycin anddexamethasone.

The use of tobramycin and dexamethasone in combination to treatophthalmic infections and attendant inflammation is known. Similarly,the use of these compounds in combination to treat inflammation andprophylactically treat (i.e., prevent or ameliorate) infections, such asin conjunction with an ocular surgical procedure, is also known. Aproduct of this type is marketed by Alcon Laboratories, Inc. in theUnited States and other countries as TOBRADEX® (tobramycin0.3%/dexamethasone 0.1%) Ophthalmic Suspension. This product has beenavailable in the United States since 1988. It has been widely acceptedas being the state-of-the-art ophthalmicanti-infective/anti-inflammatory product for many years. Further detailsregarding the composition of TOBRADEX® brand ophthalmic suspension areprovided in U.S. Pat. No. 5,149,694.

The present invention is directed to the provision of improvedtobramycin/dexamethasone compositions for topical ocular application. Inparticular, the invention is directed to the provision of compositionsthat contain xanthan gum and have a pH in the range 5 to 6. Theviscosities of the compositions at the time of manufacture and duringstorage in a container prior to use are considerably less than wouldnormally be expected based on the concentrations of xanthan gumutilized. This lowering of the viscosity prior to use is advantageousrelative to dispensing of the compositions from a dropper bottle (e.g.,DROPTAINER™, Alcon Laboratories, Inc.) or other container whenadministering the compositions to a patient. The reduction of theviscosities of the compositions at the time of manufacture and duringstorage prior to application to the eye is attributable to ionicinteractions between the tobramycin and xanthan gum which occur at a pHof 5 to 6. Those interactions, if left uncontrolled, lead to theformation of clumps of tobramycin and xanthan gum and/or precipitationof the xanthan gum. The present invention is based in part on thediscovery of formulation components and parameters that have been shownto be effective in controlling the tobramycin/xanthan gum interactions.

As indicated above, the compositions of the present invention containxanthan gum. The use of xanthan gum as a component of ophthalmiccompositions is described in U.S. Pat. No. 4,136,177; U.S. Pat. No.6,352,978; U.S. Pat. No. 6,174,524; and U.S. Pat. No. 6,261,547. The'978 patent describes the use of is xanthan gum in combination withtobramycin. It indicates that xanthan gum and tobramycin areincompatible at a pH of 5.0 to 7.8, and teaches that thisincompatibility problem can be avoided by formulating tobramycin/xanthangum compositions to have a pH in the range of 7.9 to 8.6. A productbased on the invention described in the '978 patent is marketed byaffiliates of Alcon Laboratories, Inc. in Europe and several othercountries.

The '524 and '547 patents describe xanthan-based ophthalmic compositionsformulated as non-gelled liquids that gel upon topical application tothe eye. The compositions of the '524 and '547 patents are formulated sothat their total ionic strength is approximately 120 mM or less, andpreferably about 94 mM or less. The compositions of the '524 and '547patents that have a total ionic strength greater than about 120 mM donot gel upon contact with the eye. The compositions of '524 and '547patents are generally viscous and gel upon topical application to theeye. In contrast, the compositions of the present invention generallyhave lower viscosities in the bottle, but the viscosities increasesignificantly following application to the eye, as interactions betweentobramycin and xanthan gum are broken down.

The tobramycin/dexamethasone compositions of the present invention areformulated at a pH of 5 to 6. This pH range is necessary in order tomaintain the stability of dexamethasone. The use of a pH in this rangefor an ophthalmic tobramycin/dexamethasone composition is described inU.S. Pat. No. 5,149,694. TOBRADEX® (tobramycin 0.3%/dexamethasone 0.1%)Ophthalmic Suspension also has a pH in this range.

The present invention resulted from an effort to create improvedtobramycin/dexamethasone formulations, particularly compositions thatprovide for enhanced bioavailability of tobramycin and/or dexamethasoneupon topical application to the eye, via the use of xanthan gum as avehicle for tobramycin and dexamethasone. However, as described above,it was discovered that ionic interactions between tobramycin and xanthangum at a pH of 5 to 6 lead to clumping and/or precipitation of thexanthan gum. In addition, it was discovered that xanthan gum slowlyundergoes deacetylation during storage, thereby resulting in a stabilityproblem. As explained in greater detail below, the present invention isbased on the discovery of solutions to these problems.

SUMMARY OF THE INVENTION

The present invention is directed to the provision of improvedpharmaceutical compositions that contain tobramycin and dexamethasoneand are suitable for topical application to the eyes of human patients.The compositions of the present invention are based in-part on thediscovery of formulation parameters that control ionic interactionsbetween tobramycin and xanthan gum, while maintaining the stability ofdexamethasone. The control of those interactions has enabled the presentinventors to provide compositions having physical properties that arevery advantageous. More specifically, the compositions of the presentinvention have advantageous rheological properties, as a result of thecontrolled interactions between tobramycin and xanthan gum, and thoseproperties enhance the bioavailability of drugs administered topicallyto the eye, particularly tobramycin and dexamethasone. In addition, thecompositions provide significant improvements relative to the suspensionof relatively insoluble forms of dexamethasone therein (i.e.,dexamethasone alcohol), such that even if a patient occasionally failsto comply with instructions to shake a bottle containing thecompositions prior to application to the eye, the availability ofdexamethasone suspended in the compositions is not significantlydiminished.

Solutions or suspensions containing xanthan gum at the concentrationsutilized in the present invention are normally very viscous. Asexplained in greater detail below, the present invention is based inpart on the finding that tobramycin, which is a cationic molecule,interacts ionically with the negatively charged xanthan gum molecules,thereby lowering the viscosity of the compositions. Upon application tothe eye, the viscosity of the tobramycin/xanthan gum compositions of thepresent invention is restored (i.e., increases), as a result ofdisruption of the ionic interactions between tobramycin and xanthan gum,thereby resulting in increased ocular retention and enhanced ocularbioavailability. However, during manufacture of the compositions, aswell as during storage of the compositions prior to use, the ionicinteractions between tobramycin and xanthan gum must be controlled, soas to avoid the formation of precipitates and clumping, and maintain auniform dispersion of the xanthan gum in the compositions. The presentinvention is based in-part on the identification of formulation featuresand parameters that control the ionic interaction between tobramycin andxanthan gum during the manufacturing and storage phase while maintainingthe stability of dexamethasone.

Tobramycin is a positively charged molecule, while xanthan gum isnegatively charged. When combined in an aqueous solution or suspensionat an acidic pH, the tobramycin will cause the xanthan gum toprecipitate or form clumps. Such precipitation or clumping isunacceptable in two respects. First, the tobramycin and xanthan gum areno longer uniformly distributed in the composition. This is unacceptablebecause each drop of the composition, upon dispensing from a suitablebottle or other container, must provide a uniform and predictable amountof the components of the composition, particularly the activeingredients. Second, the precipitation or clumping effect of tobramycinon xanthan gum results in a loss of the viscosity-enhancing effect ofthe xanthan gum on the composition, such that the viscosity of thecomposition may revert to a value equivalent to water (i.e., about 1centipoise).

U.S. Pat. No. 6,352,978 is based in-part on the discovery that theseionic interactions may be controlled by utilizing an alkaline pH (i.e.,a pH of 8.0 or greater). However, the use of an alkaline pH is notpossible in the tobramycin/dexamethasone compositions of the presentinvention, because dexamethasone is not stable at this pH level.Dexamethasone is stable at a pH of 5 to 6, but at this pH the negativelycharged xanthan gum and positively charged tobramycin interact to formprecipitates and/or agglomerated clumps of material.

The present inventors have discovered that the above-discussed problemscan be overcome by utilizing ionic species to control the ionicinteraction between tobramycin and xanthan gum, so as to avoid formationof precipitates or clumps and maintain the viscosity of the presenttobramycin/dexamethasone suspensions or solutions within an acceptablerange prior to application to the eye. This control is achieved viainclusion of ionic species that associate with xanthan gum ortobramycin, thereby reducing direct interactions between thesecompounds. The ionic species utilized for this purpose can be anypharmaceutically acceptable agents that dissociate into anions andcations at a pH in the range of 5 to 6, but preferably are inorganicelectrolytes or organic buffering agents, such as sodium chloride,potassium chloride or sodium sulfate.

Upon application to the eye, the viscosity of compositions of thepresent invention is restored, due to disruption of the complexesbetween xanthan gum and tobramycin. That is, upon application to theeye, the viscosity of the compositions of the present invention rises,thereby increasing the length of time during which compositions areretained on the corneal surface and enhancing ocular bioavailability.For example, as a result of this enhanced ocular bioavailability, it hasbeen determined that a dexamethasone concentration of only 0.05 w/v % inthe compositions of the present invention is bioequivalent to adexamethasone concentration of 0.1 w/v % in TOBRADEX® OphthalmicSuspension.

The present invention is also based in-part on the discovery that thexanthan gum-based compositions of the present invention possess superiorsuspension properties. More specifically, dexamethasone particles remainsuspended in the compositions of the present invention significantlylonger, relative to the prior TOBRADEX® formulation. This improvementprovides important advantages, particularly with respect to patients whosometimes forget or overlook the instructions to “shake well beforeusing” that apply to all ophthalmic suspension compositions.

The present invention is also based in-part on a finding that xanthangum is much more effective as a viscosity enhancing agent in thecompositions of the present invention if it is at least partiallydeacetylated. More specifically, xanthan gum slowly undergoesdeacetylation in aqueous solutions. It has been determined that suchdeacetylation further lowers the pH of the compositions, therebyincreasing ionic interactions between tobramycin and dexamethasone.These interactions initially result in a loss of viscosity andultimately cause clumping and/or precipitation of xanthan gum andtobramycin. The present inventors have determined that this problem canbe overcome by deacetylating xanthan gum prior to its inclusion in thecompositions of the present invention.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a graph showing the effect of sodium chloride concentration onthe viscosity of a representative formulation of the present invention,as described in Example 3;

FIG. 2 is a graph showing the effect of pH on the viscosity of arepresentative formulation of the present invention, as described inExample 3;

FIG. 3 is a graph showing the effect of a phosphate-buffered salinesolution having a pH of 7.4 on the viscosity of a representativeformulation of the present invention, as described in Example 3;

FIG. 4 is a graph illustrating the relationship between sodium chlorideequivalent concentration and viscosity, as described in Example 4; and

FIG. 5 is a graph showing ocular bioavailability data for threerepresentative formulations of the present invention, in comparison to aprior art formulation, as described in Example 5.

DESCRIPTION OF PREFERRED EMBODIMENTS

The compositions of the present invention are formulated as sterileaqueous suspensions comprising tobramycin at a concentration of 0.1 to0.5 weight/volume percent (w/v %), preferably 0.3 w/v %; dexamethasoneat a concentration of 0.03 to 0.1 w/v %, preferably 0.05 w/v %; anaqueous vehicle containing deacylated xanthan gum at a concentration of0.3 to 0.9 w/v %, preferably 0.6 w/v %; and ionic species in an amountsufficient to limit interactions between tobramycin and xanthan gum,such that the viscosity of the suspensions is maintained within therange of 10 to 700 centipoise (“cps”) preferably 10 to 300 cps, for aperiod of 18 months subsequent to the date of manufacture. Thesuspensions have a pH in the range of 5 to 6.

The ionic species utilized in the present invention can be anypharmaceutically acceptable compound that dissociates into cationic andanionic components at a pH in the range of 5 to 6. The compounds may beinorganic or organic, but will preferably be inorganic electrolytes,organic buffering agents or combinations thereof. Examples of such ionicspecies include sodium chloride, potassium chloride, calcium chloride,magnesium chloride, sodium sulfate, sodium citrate, potassium citrate,sodium phosphate, potassium phosphate, sodium acetate, sodium borate,boric acid/mannitol complexes, boric acid/sorbitol complexes andcombinations thereof.

The total amount of ionizable species present in the compositions of thepresent invention affects the viscosity of the compositions. Thecompositions must contain one or more ionizable compounds in an amountsufficient to reduce or preclude ionic interactions between tobramycinand xanthan gum, such that the formation of precipitates or clumping inthe compositions is avoided, without exceeding the viscosity rangesspecified above. The compositions therefore must contain ionic speciesin an amount sufficient to provide the compositions with a viscosity atthe time of manufacture (referred to herein as “initial viscosity”) ofat least 10 cps, preferably an amount sufficient to provide an initialviscosity of 15 cps or greater, and most preferably an amount sufficientto provide an initial viscosity of 25 cps or greater. The initialviscosity of the compositions is preferably in the range of 25 to 175cps.

The effect of ionic species on ionic strength and viscosity is dependenton the particular ionic species selected. For example, the effect ofsodium sulfate on ionic strength and viscosity is about 5.3 timesgreater than the effect of sodium chloride. The relative effect ofdifferent ionized salts maybe determined by means of routineexperimentation, within the pH range, tobramycin concentrations, xanthangum concentrations and viscosity ranges specified herein. The onlycritical parameters, so far as the compositions of the present inventionis concerned, is that the amount of ionizable salts must be sufficientto avoid formation of precipitates or clumping of tobramycin and xanthangum, without elevating the viscosity of the composition above 700 cpsor, more preferably, 300 cps.

The viscosities of the ophthalmic suspensions of the present inventionmay increase somewhat over time, due to loss of moisture from thecompositions. The suspensions are therefore formulated so as to maintainthe viscosities thereof within the range of 10 to 700 cps, preferably 10to 300 cps, over a period of 18 months. The viscosity of thecompositions of the present invention from the time of manufacture untilapplication to the eye is referred to herein as the “in vitro viscosity”of the compositions.

The viscosity values expressed herein are based on the use of aBrookfield viscometer at a shear rate of approximately 6 sec⁻¹ and at atemperature of 25° C. A shear rate of approximately 6 sec⁻¹ can beachieved using spindle CP-52 at 3 revolutions per minute “rpm”), spindleCP-51 at 1.5 rpm, spindle CP-42 at 1.5 rpm or spindle CP-41 at 3 rpm.Spindles CP-52 and CP-51 are typically used to measure viscositiesgreater than 300 centipoise (“cps”). Spindles CP-42 and CP-41 aregenerally typically used to measure viscosities less than 300 cps.

As indicated above, the viscosity of the compositions of the presentinvention is restored upon application to the eye, such that theviscosity of a composition following topical ocular administration isgreater than its viscosity while stored in a container, followingmanufacture and prior to application to the eye. This increase is causedby a shift in the pH and ionic strength of the compositions when a smallamount thereof (i.e., one or two drops) comes into contact with thelacrimal fluid of human eyes (i.e., tears). That is, the electrolytes inthe lacrimal fluid raise the pH and ionic strength of the compositions,which causes the viscosity of the compositions to increase, therebyenhancing the ocular retention and bioavailability of the compositions.

It is not readily possible to measure the viscosity of the compositionsof the present invention in vivo, i.e., following application to theeye. However the simulated in vivo viscosity model described below canbe utilized to evaluate the effect of lacrimal fluid on the viscosity ofthe compositions of the present invention in vivo. The viscosity of thecompositions of the present invention in vivo (i.e., following topicalapplication to the eye) is simulated by adding a small amount of thefollowing phosphate-buffered saline solution to the compositions:

Phosphate-Buffered Saline Solution Utilized for Simulated In VivoViscosity Measurements

Ingredient Amount (w/v %) Dibasic Sodium Phosphate 0.57% (anhydrous)Monobasic Sodium Phosphate 0.08% Monohydrate Sodium Chloride 0.65%Purified Water QS to 100% pH 7.4The addition of the above-described phosphate-buffered saline solution(“PBS solution”) to the compositions of the present invention simulatesthe effect of lacrimal fluid on the viscosity of the compositions. ThePBS solution is added to the compositions at a ratio of 1 to 10, i.e.,one part PBS solution per ten parts of thetobramycin/dexamethasone/xanthan gum compositions of the presentinvention.

For purposes of the present specification, the actual in vivo viscosityfor a composition of the present invention is presumed to be the same asthe simulated in vivo viscosity for such composition. All references to“in vivo viscosity” herein are therefore interchangeable with “simulatedin vivo viscosity”. All references herein to “simulated in vivoviscosity” and “in vitro/in vivo viscosity ratio” are based on the useof the above-described viscosity measurement procedures and simulated invivo viscosity model.

The ratio of the viscosity of a composition of the present inventionprior to application to the eye to the viscosity of the same compositionfollowing application of one drop thereof to the eye is referred toherein as the “in vitro/in vivo viscosity ratio”. The compositions ofthe present invention preferably have an in vitro/in vivo viscosityratio in the range of from 1/100 to 65/100 or 0.01 to 0.65.

The foregoing ratio may also be expressed in terms of percentages, i.e.,the in vitro viscosity divided by the simulated in vivo viscositymultiplied by 100. The foregoing range for the ratio of in vitro tosimulated in vivo viscosity is therefore equivalent to a range whereinthe in vitro viscosity of a composition of the present invention is from1% to 65% of the simulated in vivo viscosity of said composition.

The relative viscosity values may also be expressed as a ratio of invivo viscosity to in vitro viscosity. The compositions of the presentinvention preferably have an in vivo/in vitro viscosity ratio of 100/1to 100/65, which is equivalent to a range wherein the in vivo viscosityof a composition is from about 1.5 to 100 times greater than the invitro viscosity of said composition.

The tobramycin, dexamethasone and xanthan gum utilized in the sterileophthalmic suspensions of the present invention are known compounds andare readily available from various sources. A non-salt form ofdexamethasone, such as dexamethasone alcohol, is preferred. However, asalt form of dexamethasone, such as dexamethasone sodium phosphate, canalso be utilized. When a dexamethasone salt is selected, the ionicstrengths contributed by the ions formed upon dissociation of thedexamethasone salt must be considered when determining theconcentrations of ionizable species required to control the ionicinteractions between tobramycin and xanthan gum.

A pharmaceutical grade of xanthan gum should be utilized. The xanthangum should preferably be polish-filtered prior to use. The selection ofappropriate filtering techniques can be readily determined by a personskilled in the art. As discussed above, the xanthan gum must bedeacetylated, so as to enhance the stability of the suspensions of thepresent invention during storage. The acetate content of xanthan gum isbased on the acetate bound to the xanthan gum. The acetate content istypically expressed as a percent of xanthan gum, based on weight. Thexanthan gum raw material will typically have up to 6% bound acetate. Thedeacetylated xanthan gum utilized in the present invention contains lessthan 2% bound acetate, and preferably less than 1% bound acetate. Theimportance of deacetylating xanthan gum and a process by whichdeacetylation may be performed are further explained in Examples 1 and2, below.

As indicated above, the compositions of the present invention have a pHof from 5 to 6. The compositions will also have an ophthalmicallyacceptable osmolality, which is typically in the range of 200 to 400milliOsmoles per kilograms of water (mOsm/kg). When selecting bufferingagents suitable for maintaining the pH of the compositions within thespecified range of 5 to 6 and/or selecting an osmolality-adjustingagent, the impact of such agents on the ionizable species content of thecompositions must be considered. For example, if the addition of sodiumchloride for purposes of adjusting osmolality increases the ionicspecies concentration beyond a level that is acceptable (i.e., relativeto the targeted viscosity value), it may be necessary to replace all orpart of the sodium chloride with a non-ionic osmolality-adjusting agent,such as propylene glycol.

The compositions of the present invention may contain various otheringredients that are typically utilized in ophthalmic pharmaceuticalcompositions, such as antimicrobial preservatives (e.g., benzalkoniumchloride) and wetting agents (e.g., tyloxapol and Polysorbate 80). Thecompositions are preferably formulated and packaged as multi-doseproducts, but may also be formulated without a conventionalantimicrobial preservative and packaged in a sealed, unit dose vial.

The compositions of the present invention are useful in the treatment ofocular inflammatory conditions wherein either an infection or a risk ofinfection exists. As utilized herein, the term “treatment” encompassesboth active treatment of an existing condition and prophylactictreatment of a patient that is at risk of developing a condition (e.g.,infection). The compositions of the present invention are particularlyuseful in treating ocular inflammation associated with injuries to theeye resulting from trauma, as well as inflammation associated withocular surgical procedures (e.g., cataract surgery, retinal surgery,LASIK surgery) and ocular injections (e.g., retrobulbar injections,posterior juxtascleral injections and anterior juxtascleral injections).

Such treatments can be performed by applying a small amount (e.g., oneto two drops) of a composition of the present invention to the affectedeye or eyes of a patient from two to four times per day. However, boththe amount of the dose and the dosing frequency may be modified byclinicians.

Example 1

The preparation of tobramycin/dexamethasone/xanthan gum formulationsutilizing xanthan gum that has not bee deacetylated is described below.The stability of the resulting formulations was also evaluated, asexplained below.

Preparation of Xanthan Gum Stock Solution

Hot water was added to a vessel. Xanthan gum was weighed and slowlyadded to the vessel while mixing. The temperature was adjusted to 60° C.and the xanthan gum and water were mixed until uniform. Purified waterwas added to bring the composition to the final target weight and mixeduntil uniform. The temperature was increased to 70° C. prior tofiltering through an appropriate polishing filter e.g., 1.2 um filter.

Preparation of Tobramycin/Dexamethasone Formulations Using Xanthan GumStock Solution

The amounts of tobramycin, sodium chloride, boric acid and disodiumedetate specified in Table 1A below were added to a portion of thepurified water and dissolved. Hydrochloric or sulfuric acid was added toreduce pH. Tyloxapol and dexamethasone were added as slurry or aspowder. Batch quantity of xanthan gum stock solution was added and mixedwell. 1N hydrochloric acid or 1N sulfuric acid were added to reach thetarget pH. Purified water was added to bring to final volume and mixedwell. The viscosities of the resulting formulations were measured at ashear rate of 6 sec⁻¹. The respective viscosity values are shown inTable 1A below.

TABLE 1A Formulation Number 107201 107209 W/V % W/V % INGREDIENTSTobramycin 0.3 0.3 Dexamethasone 0.1 0.1 Xanthan Gum 0.9 0.9 Sodiumchloride 0.42 0.08 Tyloxapol 0.05 0.05 Boric Acid 0.5 1 Disodium Edetate0.01 0.01 Sodium Hydroxide Adjust pH Adjust pH to 5.5 to 5.7Hydrochloric Acid Adjust pH None to 5.5 Sulfuric Acid None Adjust pH to5.7 Purified Water Qs to 100% Qs to 100% RESULTS Viscosity at shear 418642 rate 6 sec−1 (cps)

The formulations described in Table 1A were subjected to acceleratedstability testing. As shown in Table 1B, below, the pH and viscositiesof the formulations, which were prepared using xanthan gum that has notbeen deacetylated, decrease upon storage. This eventually makes theformulations unusable. Specifically, the uniform nature of thesuspensions was lost.

TABLE 1B Stability of pH and Viscosity of Tobramycin/DexamethasoneFormulations Prepared Using Non-Deacetylated Xanthan Gum FormulationNumber 107201 107209 107201 107209 Analysis pH Viscosity of Formulation(cps) Initial 5.48 5.74 418 642 40° C., 4 Weeks 5.33 5.56 187 217 40°C., 8 Weeks 5.08 5.36 86 141 40° C., 16 Weeks 4.86 4.89 25 37 50° C., 1Week 5.37 5.73 175 240 50° C., 2 Weeks 5.20 5.25 95 160 50° C., 4 Weeks5.10 5.14 48 91 50° C., 8 Weeks 4.70 4.81 Not Not Uniform Uniform 60°C., 1 Week 5.20 5.16 68 132 60° C., 2 Weeks Not 4.83 Not 43 UniformUniform 60° C., 4 Weeks Not Not Not Not Uniform Uniform Uniform Uniform

Example 2

The preparation of tobramycin/dexamethasone formulations in accordancewith the principles of the present invention, including the use ofdeacetylated xanthan gum, is described below.

Preparation of Xanthan Gum Stock Solution and Pretreatment with Base

Hot water was added to a vessel. Xanthan gum was weighed and slowlyadded to the vessel while mixing. 2.5 ml of 1 N NaOH or equivalent per 1g of xanthan gum was added and then mixed for 20 minutes. 1.66 ml of 1NHCl or equivalent per 1 g of xanthan gum was then added. Purified waterwas added to adjust the target weight followed by mixing for 15 minutes.The deacetylated xanthan gum was then filtered through an appropriatefilter e.g., 1.2 um filter.

Preparation of a Tobramycin/Dexamethasone Formulation Using Pre-TreatedXanthan Gum Stock Solution

The specified amounts of tobramycin, sodium chloride, sodium sulfate,disodium edetate, and propylene glycol were added to a portion of thepurified water, following by addition of tyloxapol and dexamethasone asa slurry or as powder. The pH was adjusted using 1 N hydrochloric acidto a pH slightly higher than the target pH. The deacetylated xanthan gumstock solution described above was then added and the resultingsuspension was mixed well. The pH was adjusted with HCl and/or NaOHsolution to the target level and the viscosity of the formulation wasmeasured.

TABLE 2A Formulation Number 108536 W/V % INGREDIENTS Tobramycin 0.3Dexamethasone 0.1 Xanthan Gum 0.6 Sodium chloride 0.24 Propylene Glycol0.6 Tyloxapol 0.05 Sodium Sulfate (Anhydrous) 0.25 Disodium Edetate 0.01Benzalkonium Chloride 0.01 Sodium Hydroxide Adjust pH to 5.75Hydrochloric Acid Adjust pH to 5.75 Purified Water Qs to 100% RESULTSViscosity at shear 116 rate 6 sec−1 (cps) Simulated In Vivo 1059Viscosity at shear rate 6 sec−1 (cps) Viscosity of Formulation 11% as a% of Simulated In Vivo ViscosityAs shown in Table 2B, below, the pH values for Formulation 108536, whichcontains deacetylated xanthan gum, were fairly stable upon storage,unlike that of Formulations 107201 and 107209 in Example 1. As a result,the viscosities of Formulation 108536 remained stable or increasedduring storage, rather than decreasing, as in Example 1.

TABLE 2B Stability of pH and Viscosity of Tobramycin/DexamethasoneFormulations Prepared Using Deacetylated (Pre-treated) Xanthan GumFormulation Number 108536 Analysis pH Pre-dose Viscosity (cps) Initial5.84 116 40° C., 4 Weeks 5.80 166 40° C., 8 Weeks 5.81 167 40° C., 12Weeks 5.81 181 40° C., 16 Weeks ND ND 40° C., 26 Weeks ND ND 50° C., 1Week ND ND 50° C., 2 Weeks 5.79 152 50° C., 4 Weeks 5.78 179 50° C., 8Weeks 5.76 271 50° C,. 12 Weeks 5.73 372 50° C., 16 Weeks ND NA 60° C.,1 Week 5.79 150 60° C., 2 Weeks 5.78 172 60° C., 3 Weeks ND ND 60° C., 4Weeks 5.66 235 ND = Not Determined

Example 3

The effect of tobramycin on the initial viscosity of the compositions ofthe present invention and the recovery of viscosity upon application ofthe compositions to the eye are further illustrated herein. Theformulation shown in Table 3A below, which is a different lot ofFormulation Number 108536 described in Table 2A above and isrepresentative of the compositions of the present invention, wasprepared utilizing deacetylated xanthan gum. The initial viscosity ofthe formulation was measured at a shear rate of 6 sec⁻¹ and determinedto be 42 cps.

TABLE 3A Component % w/v Function Tobramycin 0.3 Active Dexamethasone(Micronized) 0.1 Active Benzalkonium Chloride 0.01 PreservativeTyloxapol 0.05 Wetting Agent Disodium Edetate 0.01 Preservation AidSodium Chloride 0.23 ± 0.04 Tonicity Agent Sodium Sulfate 0.25 TonicityAgent Xanthan Gum 0.6% Viscosity Modifier Propylene Glycol 0.6 TonicityAgent Hydrochloric Acid and/or Adjust pH pH Adjustment Sodium Hydroxideto 5.7 Purified Water QS 100 Vehicle

A second formulation, which was identical to the formulation shown inTable 3A, except for the omission of tobramycin, was also prepared. Thesecond formulation was determined to have an initial viscosity of 836cps.

A slight increase in pH or addition of small amount of ions (e.g. sodiumchloride, phosphate buffer) reduces the ionic interactions betweentobramycin and xanthan gum, thereby restoring the formulation viscosity.This phenomenon is graphically presented in FIGS. 1-3. FIG. 1 shows thatthe viscosity of the formulation described in Table 3A increases from 42cps to over 1,000 cps upon addition of 0.2 g of sodium chloride to 100mL of the formulation. FIG. 2 shows that the viscosity of theformulation increases from 42 cps at pH 5.7 to over 1,100 cps is when pHis adjusted upward to 6.2, and to 1,300 cps when pH is at 6.4. FIG. 3shows that the viscosity of the formulation increases from 42 cps to1,059 cps upon addition of 10 mL of the above-described PBS solution to100 mL of the suspension.

When tobramycin was removed from the formulation shown in Table 3A, theviscosity of the formulation did not increase after mixing with the PBSsolution. Specifically, a modified version of the formulation, withouttobramycin, was determined to have a viscosity of 667 cps when 10 ml ofphosphate buffered saline solution was added to 100 ml of theformulation. In other words, the viscosity of the modified formulationwas actually reduced from an initial viscosity of 836 cps to a simulatedin vivo viscosity of 667 cps, following addition of the phosphatebuffered saline solution.

Example 4

As discussed and illustrated below, the viscosity of the compositions ofthe present invention is affected by the ionic strength of thecompositions and pH, as well as the amounts of tobramycin and xanthangum selected within the specified ranges of 0.1 to 0.5 w/v % and 0.3 to0.9 w/v %, respectively. The formulations and associated data presentedin Tables 4A-4E are provided to further illustrate and explain theinteraction of these factors.

A comparison of Formulations A-D and the respective viscosity values foris these compositions illustrates the impact of tobramycin on theviscosity of a composition containing xanthan gum at a concentration of0.6 w/v %. Specifically, Formulation A, which contains tobramycin at aconcentration of 0.3 w/v %, has an initial viscosity of 15 centipoise(“cps”), while Formulation C, which is identical to Formulation A exceptfor the absence of tobramycin, has an initial viscosity of 919 cps.Thus, the presence of tobramycin in Formulation A contributes to thelowering of the viscosity of the composition. This effect of tobramycinis also evident based on a comparison of Formulations B and D.(Formulations A and B do not contain dexamethasone, but are otherwiserepresentative examples of the tobramycin/dexamethasone compositions ofthe present invention. Formulations C and D are provided for comparativepurposes and are not representative examples of the compositions of thepresent invention.)

The viscosity of Formulation A is stabilized by the inclusion of 23.9 mM(0.34%) of sodium sulfate, which is a preferred ionizable species.Formulation A also includes about 10 mM of sodium chloride, asdeacetylated xanthan gum stock solution contains sodium chloride, formedby the addition of sodium hydroxide and hydrochloric acid during thedeacetylation step. The ionic contributions from EDTA (disodium edetate)and benzalkonium chloride are insignificant, as their concentrations arevery low.

The viscosity of Formulation B is stabilized by the inclusion of 138.2mM sodium chloride, which is also a preferred ionizable species.

The viscosity of the compositions of the present invention can bestabilized using sodium chloride or sodium sulfate. However theconcentration of sodium sulfate required is much smaller than theconcentration of sodium chloride. Approximately 1 mM of sodium sulfateis equivalent to 5.3 mM of sodium chloride. This is demonstrated byExamples A, B and E though L.

The viscosities of Formulations A, B and E though L versus sodiumchloride equivalent ionic concentration is plotted in FIG. 4. The sodiumchloride equivalent ionic concentration for these formulations isdefined as “sodium chloride concentration (mM)+5.3 sodium sulfateconcentration (mM)”. The viscosities of the formulations containing 0.3%tobramycin and 0.6% xanthan gum increases as the sodium chlorideequivalent ionic concentration increases. The viscosity is in thepreferred range of 10 to 300 cps for sodium chloride equivalent ionicconcentration range of 134 to 150 mM.

Other ionizable species can be used in place of sodium chloride orsodium sulfate. The preferred ionized salts are sodium chloride, sodiumsulfate, sodium citrate, sodium phosphate, sodium borate, sodiumacetate, potassium chloride, calcium chloride, and magnesium chloride.The different ionized species will need a different factor (which is 5.3for sodium sulfate) to determine the sodium chloride equivalentconcentration. This factor can be determined by making samples withdifferent ratios of sodium chloride and the other salt. The viscosityresults of those samples can then be analyzed to determine the factorfor determining the sodium chloride equivalent concentration. Thisfactor will be greater than one for salts with multivalent ions.

For a given active moiety and its concentration, the sodium chlorideequivalent ionic concentration range that provides relatively lowviscosity depends on pH and xanthan gum concentration. For a 0.3%tobramycin solution, Formulations M and N show that a higher sodiumchloride equivalent ionic concentration is required to provide thesimilar viscosity at lower pH of 5.5 compared to that at pH of 5.75.

Formulations O, P and Q show that at a fixed pH (5.5), lower sodiumchloride equivalent ionic concentrations are required as xanthan gumconcentration is increased from 0.6% to 0.9%.

TABLE 4A Formulation A B C D INGREDIENTS W/V % W/V % W/V % W/V %Tobramycin 0.3 0.3 None None Xanthan Gum 0.6 0.6 0.6 0.6 Sodium Chloride0 0.75 0 0.75 Sodium Sulfate 0.34 0 0.34 0 Tyloxapol 0.05 0.05 0.05 0.05Disodium Edetate 0.01 0.01 0.01 0.01 Benzalkonium Chloride 0.01 0.010.01 0.01 Propylene Glycol 0.6 0.6 0.6 0.6 Hydrochloric Acid Adjust pHAdjust pH Adjust pH Adjust pH to 5.7 to 5.7 to 5.7 to 5.7 SodiumHydroxide Adjust pH Adjust pH Adjust pH Adjust pH to 5.7 to 5.7 to 5.7to 5.7 Purified Water Qs to 100% Qs to 100% Qs to 100% Qs to 100% SodiumChloride from 10.0 10.0 10.0 10.0 Xanthan Stock, mM Sodium Chloride 0.0128.2 0.0 128.2 added, mM Total Sodium Chloride 10.0 138.2 10.0 138.2Concentration (mM) Sodium Sulfate 23.9 0.0 23.9 0.0 Concentration (mM)Sodium Chloride 137 138 137 138 concentration (mM) + 5.3 Sodium SulfateConcentration (mM) Viscosity at shear 15 22 919 915 rate 6 sec−1 (cps)Simulated In Vivo 783 817 786 786 Viscosity at shear rate 6 sec−1 (cps)Viscosity of Formulation 2% 3% 117% 116% as a % of Simulated In VivoViscosity

TABLE 4B Formulation E F G H 12076: 14I 12076: 17Q 12076: 14J 12076: 14KINGREDIENTS W/V % W/V % W/V % W/V % Tobramycin 0.3 0.3 0.3 0.3 XanthanGum 0.6 0.6 0.6 0.6 Sodium Chloride 0.1 0.55 0.23 0.35 Sodium Sulfate0.3 0.1 0.25 0.2 Tyloxapol 0.05 0.05 0.05 0.05 Disodium Edetate 0.010.01 0.01 0.01 Benzalkonium Chloride 0.01 0.01 0.01 0.01 PropyleneGlycol 0.6 0.6 0.6 0.6 Hydrochloric Acid Adjust pH Adjust pH Adjust pHAdjust pH to 5.7 to 5.7 to 5.7 to 5.7 Sodium Hydroxide Adjust pH AdjustpH Adjust pH Adjust pH to 5.7 to 5.7 to 5.7 to 5.7 Purified Water Qs to100% Qs to 100% Qs to 100% Qs to 100% Sodium Chloride from 10.0 10.010.0 10.0 Xanthan Stock, mM Sodium Chloride added, mM 17.1 94.0 39.359.8 Total Sodium Chloride 27.1 104.0 49.3 69.8 Concentration (mM)Sodium Sulfate 21.1 7.0 17.6 14.1 Concentration (mM) Sodium Chloride 139141 143 144 concentration (mM) + 5.3 Sodium Sulfate Concentration (mM)Viscosity at shear 40 58 83 83 rate 6 sec−1 (cps) Simulated In Vivo 857823 836 823 Viscosity at shear rate 6 sec−1 (cps) Viscosity ofFormulation 5% 7% 10% 10% as a % of Simulated In Vivo Viscosity

TABLE 4C Formulation I J K L INGREDIENTS W/V % W/V % W/V % W/V %Tobramycin 0.3 0.3 0.3 0.3 Xanthan Gum 0.6 0.6 0.6 0.6 Sodium Chloride0.45 0.5 0.6 0 Sodium Sulfate 0.15 0.15 0.1 0.4 Tyloxapol 0.05 0.05 0.050.05 Disodium Edetate 0.01 0.01 0.01 0.01 Benzalkonium Chloride 0.010.01 0.01 0.01 Propylene Glycol 0.6 0.6 0.6 0.6 Hydrochloric Acid AdjustpH Adjust pH Adjust pH Adjust pH to 5.7 to 5.7 to 5.7 to 5.7 SodiumHydroxide Adjust pH Adjust pH Adjust pH Adjust pH to 5.7 to 5.7 to 5.7to 5.7 Purified Water Qs to 100% Qs to 100% Qs to 100% Qs to 100% SodiumChloride from 10.0 10.0 10.0 10.0 Xanthan Stock, mM Sodium Chlorideadded, mM 76.9 85.5 102.6 0.0 Total Sodium Chloride 86.9 95.4 112.5 10.0Concentration (mM) Sodium Sulfate 10.6 10.6 7.0 28.2 Concentration (mM)Sodium Chloride 143 151 150 159 concentration (mM) + 5.3 Sodium SulfateConcentration (mM) Viscosity at shear 95 433 547 1016 rate 6 sec−1 (cps)Simulated In Vivo 823 811 820 854 Viscosity at shear rate 6 sec−1 (cps)Viscosity of Formulation 12% 53% 67% 119% as a % of Simulated In VivoViscosity

TABLE 4D Formulation M N Batch No. 05-39669 05-39450 INGREDIENTS W/V %W/V % Tobramycin 0.3 0.3 Dexamethasone 0.1 0.1 Xanthan Gum 0.6 0.6Sodium chloride 0.24 0.36 Sodium Sulfate 0.25 0.25 Propylene Glycol 0.60.5 Tyloxapol 0.05 0.05 Boric Acid None None Disodium Edetate 0.01 0.01Sodium Hydroxide Adjust pH Adjust pH to 5.75 to 5.5 Hydrochloric AcidAdjust pH Adjust pH to 5.75 to 5.5 Purified Water Qs to 100% Qs to 100%Sodium Chloride from 10.0 10.0 Xanthan Stock, mM Sodium Chloride added,mM 41.0 61.5 Total Sodium Chloride 51.0 71.5 Concentration (mM) SodiumSulfate 17.6 17.6 Concentration (mM) Sodium Chloride 144 165concentration (mM) + 5.3 Sodium Sulfate Concentration (mM) Viscosity atshear 116 151 rate 6 sec−1 (cps) Simulated In Vivo 1059 977 Viscosity atshear rate 6 sec−1 (cps) Viscosity of Formulation 11% 15% as a % ofSimulated In Vivo Viscosity

TABLE 4E Formulation O P Q INGREDIENTS W/V % W/V % W/V % Tobramycin 0.30.3 0.3 Dexamethasone 0.1 0.1 0.1 Xanthan Gum 0.6 0.8 0.9 Sodiumchloride 0.36 0.23 0.1 Sodium Sulfate 0.25 0.25 0.25 Propylene Glycol0.5 0.5 None Tyloxapol 0.05 0.05 0.05 Boric Acid None None 0.5 DisodiumEdetate 0.01 0.01 None Sodium Hydroxide Adjust pH Adjust pH Adjust pH to5.5 to 5.5 to 5.5 Hydrochloric Acid Adjust pH Adjust pH Adjust pH to 5.5to 5.5 to 5.5 Purified Water Qs to 100% Qs to 100% Qs to 100% SodiumChloride from 10.0 13.3 14.9 Xanthan Stock, mM Sodium Chloride added, mM61.5 39.3 17.1 Total Sodium Chloride 71.5 52.6 32.0 Concentration (mM)Sodium Sulfate 17.6 17.6 17.6 Concentration (mM) Sodium Chloride 165 146125 concentration (mM) + 5.3 Sodium Sulfate Concentration (mM) Viscosityat shear 151 163 636 rate 6 sec−1 (cps) Simulated In Vivo 977 1554 2208Viscosity at shear rate 6 sec−1 (cps) Viscosity of Formulation 15% 11%29% as a % of Simulated In Vivo Viscosity

Example 5 Rabbit Bioavailability Study Results

The ocular bioavailability of three representative compositions of thepresent invention was evaluated relative to TOBRADEX® (tobramycin0.3%/dexamethasone 0.1%) Ophthalmic Suspension. The formulations of thecompositions of the present invention are shown in Table 5A, below. Theformulation of TOBRADEX® Ophthalmic Suspension is shown in Example 1 ofU.S. Pat. No. 5,149,694.

TABLE 5A Formulation Number 109443 109442 108536 W/V % W/V % W/V %INGREDIENTS Tobramycin 0.3 0.3 0.3 Dexamethasone 0.01 0.05 0.1 XanthanGum 0.6 0.6 0.6 Sodium chloride 0.21 0.21 0.24 Propylene Glycol 0.6 0.60.6 Sodium Sulfate (Anhydrous) 0.25 0.25 0.25 Tyloxapol 0.05 0.05 0.05Disodium Edetate 0.01 0.01 0.01 Benzalkonium Chloride 0.01 0.01 0.01Sodium hydroxide pH 5.75 pH 5.75 pH 5.75 Hydrochloric acid pH 5.75 pH5.75 pH 5.75 Purified Water 100% 100% 100% RESULTS Viscosity at shear 2932 130 rate 6 sec−1 (cps) Simulated In Vivo 872 872 955 Viscosity atshear rate 6 sec−1 (cps) Viscosity of Formulation  3%  4%  14% as a % ofSimulated In Vivo Viscosity

The respective compositions were administered to both eyes of male NewZealand rabbits. Following administration of the formulations, aqueoushumor samples were collected from both eyes at 0.5, 0.75, 1, 2, and 3hours and concentrations of dexamethasone were determined using theLC-MS/MS procedure described below.

Concentrations of dexamethasone in the rabbit aqueous humor weremeasured using a validated HPLC tandem mass spectrometry (HPLC/MS/MS)method. In this procedure, a 25.0 microliter aliquot of aqueous humor isspiked with beclomethasone as internal standard and extracted usingmethyl-t-butyl ether. The organic layer is evaporated to dryness andreconstituted in 20:80 10 mM ammonium formate:methanol and injected on areversed-phase HPLC column under isocratic conditions with a mobilephase of the same composition as used for sample reconstitution. Thecolumn effluent is subjected to positive ion electrospray ionization andthe protonated molecular ions of dexamethasone and beclomethasonesubjected to collisional fragmentation. Multiple reaction monitoring ofthe m/z 393.1→373.4 and 409.3→391.4 transitions for dexamethasone andbeclomethasone, respectively, allows for specific detection. The workingrange of the procedure is 1.00 to 200 ng/mL.

Mean aqueous humor concentrations for dexamethasone versus time areplotted in FIG. 5. The maximum concentrations (C_(max)) of dexamethasonein the aqueous humor and area under the curve (AUC) values are providedin Table 5B, below;

TABLE 5B AUC_(0-3 h) Formulation C_(max) (ng/mL) (ng*h/mL) TOBRADEX ®(tobramycin 69.4 ± 21.6 118 ± 6  0.3%/dexamethasone 0.1% OphthalmicSuspension) Formulation 109443 45.6 ± 16.6 103 ± 9  (tobramycin 0.3%/dexamethasone 0.01%) Formulation 109442 106 ± 19  191 ± 10 (tobramycin0.3%/ dexamethasone 0.05%) Formulation 108536 129 ± 36  291 ± 14(tobramycin 0.3%/ dexamethasone 0.1%)

The foregoing results show that the aqueous humor concentrations for thexanthan-based formulations of the present invention, containingdexamethasone at concentrations of 0.05% and 0.1%, respectively, aremuch higher than those for TOBRADEX® Suspension, which contains 0.1%dexamethasone. These results demonstrate the superior bioavailability ofthe compositions of the present invention.

1. A topical ophthalmic composition, comprising: 0.1 to 0.5 w/v %tobramycin; 0.03 to 0.1 w/v % of dexamethasone; an aqueous,ophthalmically acceptable vehicle containing deacetylated xanthan gum ata concentration of 0.3 to 0.9%; and one or more ionizable species in anamount sufficient to limit ionic interactions between the tobramycin andthe deacetylated xanthan gum, such that the in vitro is viscosity of thecomposition is maintained within the range of 10 to 700 cps at a shearrate of 6 sec⁻¹ and a temperature of 25° C.; said composition having apH of 5 to
 6. 2. A composition according to claim 1, wherein the one ormore ionizable species is selected from the group consisting ofinorganic electrolytes, organic buffering agents and combinationsthereof.
 3. A composition according to claim 2, wherein the one or moreionizable species is selected from the group consisting of sodiumchloride, potassium chloride, calcium chloride, magnesium chloride,sodium sulfate, sodium citrate, potassium citrate, sodium phosphate,potassium phosphate, sodium acetate, sodium borate, boric acid/mannitolcomplexes, boric acid/sorbitol complexes and combinations thereof.
 4. Acomposition according to claim 3, wherein the ionizable species isselected from the group consisting of sodium chloride, sodium sulfate,and combinations thereof.
 5. A composition according to claim 1, whereinthe in vitro viscosity of the composition is within the range of 10 to300 cps.
 6. A composition according to claim 5, wherein the compositionhas an initial viscosity of 25 to 175 cps.
 7. A composition according toclaim 6, wherein the composition has an in vitro/in vivo viscosity ratioof 0.01 to 0.65.
 8. A composition according to claim 7, wherein the oneor more ionizable species is selected from the group consisting ofinorganic electrolytes, organic buffering agents and combinationsthereof.
 9. A composition according to claim 8, wherein the one or moreionizable species is selected from the group consisting of sodiumchloride, potassium chloride, calcium chloride, magnesium chloride,sodium sulfate, sodium citrate, potassium citrate, sodium phosphate,potassium phosphate, sodium acetate, sodium borate, boric acid/mannitolcomplexes, boric acid/sorbitol complexes and combinations thereof.
 10. Acomposition according to claim 9, wherein the ionizable species isselected is from the group consisting of sodium chloride, sodiumsulfate, and combinations thereof.
 11. A composition according to anyone of claims 1-10, wherein the composition contains tobramycin at aconcentration of 0.3 w/v %, xanthan gum at concentration of 0.6 w/v %and dexamethasone at a concentration of 0.05 w/v %.
 12. A compositionaccording to claim 11, wherein the pH of the composition is about 5.7.13. A method of treating ophthalmic inflammatory conditions whereineither an infection or risk of infection exists, which comprisestopically applying a therapeutically effective amount of a compositionaccording to claim 1 to the affected eye.
 14. A method of treatingophthalmic inflammatory conditions wherein either an infection or riskof infection exists, which comprises applying a therapeuticallyeffective amount of a composition according to claim 11 to the affectedeye.